Megakaryocytes (Mks) are derived from hematopoietic stem cells. Mk differentiation and maturation progresses through several overlapping stages: multiple rounds of endomitosis to form polyploid cells;development of a demarcation membrane system;an apoptotic-death program, which leads to cell disintegration;and formation of cytoplasmic extensions called proplatelets, from which platelets are released. The mechanisms governing Mk commitment, differentiation, polyploidization, and apoptosis remain poorly understood. Increasing Mk ploidy is important because the number of platelets produced increases with Mk DNA content. Ex vivo generation in bioreactors of functional platelets would have a major impact in transfusion medicine. Platelet transfusions are used for a wide range of thrombotic deficiencies and several million units are transfused each year. Platelets are an expensive product in limited supply due to the collection and processing steps from donated blood and the fact that platelets cannot be stored frozen. Rather they are stored for 3-5 days at 20-240C, which increases the risk of bacterial contamination. Blood-borne pathogens also pose a risk, and alloimmunization of recipients remains a problem. Culture-derived platelets, produced under using Good Manufacturing Practices, could provide a safer and more tolerated supply for transfusion therapies. Improved understanding and the ability to control Mk maturation will be critical for providing tissue engineering solutions that make it economically feasible to produce platelets in large scale for transfusion medicine. Several groups have generated small quantities of culture-derived platelets with functional activity similar to that of harvested platelets. However, producing even the platelets required for a single transfusion presents a major technological challenge. Major advances are needed for large-scale, culture-derived platelet production to become economically attractive. This will require improvements in the expansion of human hematopoietic and progenitor (HSPCs) cells into Mks, but also the ability to produce large, polyploid Mk cells, since the number of platelets produced from an Mk cell is proportional to the cell's ploidy. Breakup of polyploidy Mk cells takes place largely in the BM vasculature whereby Mk cells project cytoplasmic extensions through the gaps of sinus walls. Mks can also go into circulation and mature to produce platelets in the lung vasculature. Mk-cell breakup is associated with mechanical stresses due to blood flow and/or cell deformation. Thus, Mk breakup and possibly maturation is a stress-induced process. In view of the fact that Mk maturation and proplatelet formation are also affected by interactions with extracellular matrix, platelet production will need to engage bioreactor systems involving semi-synthetic matrices under flow conditions to simulate, to the extent possible, the in vivo conditions. Here we focus on the aspects of Mk maturation affected by exogenous mechanical (shear) stress. Our in vivo and ex vivo preliminary data suggest that the tumor suppressor p53 is specifically activated upon initiation of Mk differentiation. Our central hypothesis is that p53's role is to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. In this model, p53 is called to play the role of Mk ploidy regulator, and in this sense we hypothesize that this role is to respond to endogenous (due to polyploidization) and exogenous (shear) stress. Understanding the role of p53 as a transducer of these stresses, but also as a possible regulator of Mk maturation will be important in the development of scalable processes for platelet production. Using a parallel-plate flow apparatus, we aim to understand how the level of shear stress, in combination with the length of exposure to flow, affect cell cycling, endomitosis, apoptosis and p53 activation (as captured by specific acetylation/deacetylation events) in cultured human Mk cells. Microarray analysis will examine the impact of fluid forces on the gene expression patterns during Mk maturation aiming to identify the programs and genes affected. PUBLIC HEALTH RELEVANCE: Megakaryocytes (Mks) are derived from hematopoietic (blood) stem cells, and are distinguished by their very large size, high DNA content, and the formation of proplatelet extensions which shed platelets, the small cells necessary for blood coagulation. Proplatelet formation takes place in the bone marrow vasculature under mechanical stress conditions, which are essential for the platelet production. Elucidating the mechanisms responsible for Mk maturation and proplatelet formation is important for understanding the process that leads to platelet production. This could lead to identification of factors and culture conditions that promote the generation of many, high-ploidy Mk cells that would enable the production, in bioreactors, of large numbers of platelets for medical transfusions. Design of large-scale processes for achieving this goal requires that we understand and mimic to the extent possible the complex process of in vivo proplatelet formation, and this is an important goal of this project.